tail-vein-transection-bleeding-model-fully-anesthetized-hemophilia

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) ...

Tissue factor (TF) is believed to play an important role in coagulation, inflammation, angiogenesis and wound healing as well as in tumor growth and metastasis. To facilitate in vivo studies in experimental murine models, we have produced recombinant murine factor VII (FVII) and the ectodomain of murine TF, TF(1-223). Murine FVII was activated to FVIIa with human factor Xa and upon reaction with FFR-chloromethyl ketone converted into an active site-blocked TF antagonist, FFR-FVIIa. The activity of murine FVIIa was characterized in factor X activation assays as well as in clot assays with murine and human thromboplastin in murine and human plasma. In these assays murine FVIIa exhibited a specific activity equivalent to or higher than human FVIIa. Further analysis showed that murine FVIIa binds with high affinity to both murine and human TF, whereas the association of human FVIIa to murine TF is about three orders of magnitude weaker than the association to human TF. This difference was further emphasized by the effect of murine-and human FFR-FVIIa on bleeding in an in vivo mouse model. Intra-peritoneal administration of 1 mg/kg murine FFR-FVIIa significantly prolonged the tail-bleeding time, whereas no effect on bleeding was observed with a 25-times higher dose of the human FFR-FVIIa. Together, these data confirms the notion of poor species compatibility between human FVII and murine TF and emphasizes the requirement for autologous FVIIa in studies on the role of the TF in experimental in vivo pharmacology.

Characterization of recombinant murine factor VIIa and recombinant murine tissue factor: a human-murine species compatibility study.

Gewebe-Faktor (TF) ist vermutlich eine wichtige Rolle in der Gerinnung, Entz&uuml;ndung, Angiogenese und die Wundheilung ebenfalls wie bei Tumorwachstum und Metastasierung. In-vivo-Untersuchungen in murinen Versuchsmodelle zu erleichtern, haben wir rekombinante murine Faktor VII (FVII) und die APPs&alpha; des murinen TF, TF(1-223) produziert. Murine FVII wurde zu FVIIa mit menschlichen Faktor Xa und durch Reaktion FFR-Chlormethyl-Keton umgebaut eine aktive Website blockiert TF Antagonist, FFR-FVIIa aktiviert. Die T&auml;tigkeit des murinen FVIIa zeichnete sich bei Faktor X-Aktivierung-Assays sowie bei Blutgerinnsel-Assays mit murinen und menschliches Thromboplastin in murinen und menschlichen Plasma. Diese Proben stellte murinen FVIIa eine bestimmte Aktivit&auml;t gleichwertig oder h&ouml;her als menschliche FVIIa. Weitere Analyse zeigte, dass murinen FVIIa mit hoher Affinit&auml;t zu sowohl menschlich als auch murinen TF bindet, w&auml;hrend die Verbindung von menschlichen FVIIa zu murinen TF etwa drei Gr&ouml;&szlig;enordnungen schw&auml;cher als der Verein, um menschliche TF ist. Dieser Unterschied wurde weiter durch die Wirkung der Murine betont- und menschlichen FFR-FVIIa auf Blutungen in einem in-vivo Mausmodell. Intra-Peritoneal Verwaltung von 1 mg/kg Murine FFR-FVIIa verl&auml;ngert erheblich den Schwanz-Blutungen Zeit, w&auml;hrend keine Auswirkung auf die Blutung mit einer 25-mal h&ouml;here Dosis von der menschlichen FFR-FVIIa beobachtet wurde. Zusammen, diese Daten best&auml;tigt das Konzept der Armen Arten Kompatibilit&auml;t zwischen menschlichen FVII und murinen TF und betont das Erfordernis einer autologen FVIIa in Studien &uuml;ber die Rolle der TF in experimentelle in-vivo-Pharmakologie.

Charakterisierung der rekombinante murine Faktor VIIa und rekombinante murine Gewebe Faktor: ein Mensch-Murine Arten-Vertr&auml;glichkeitsstudie.

It has earlier been demonstrated that capsaicin-induced desensitization improves insulin sensitivity in normal rats. However, whether increased capsaicin-sensitive nerve activity precedes the onset of insulin resistance in diet-induced obesity--and therefore might be involved in the pathophysiology--is not known. Further, it is of relevance to investigate whether capsaicin desensitization improves glycaemic control even in obese individuals and we therefore chose the obese Zucker rats to test this.

Plasma calcitonin gene-related peptide is increased prior to obesity, and sensory nerve desensitization by capsaicin improves oral glucose tolerance in obese Zucker rats.

Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the K(m) for activation by FVIIa-tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood-based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

Article	Year
Characterization of recombinant murine factor VIIa and recombinant murine tissue factor: a human-murine species compatibility study. Thrombosis research\| PubMed ID: 15850611	2005
Plasma calcitonin gene-related peptide is increased prior to obesity, and sensory nerve desensitization by capsaicin improves oral glucose tolerance in obese Zucker rats. European journal of endocrinology\| PubMed ID: 16322403	2005
Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide. Blood\| PubMed ID: 21700771	2011

Torben Elm

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